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Centro De Ciencias Básicas Departamento De Fisiología CENTRO DE CIENCIAS BÁSICAS DEPARTAMENTO DE FISIOLOGÍA Y FARMACOLOGÍA TESIS CARACTERIZACIÓNBIOQUÍMICA DEL VENENO DETRES SERPIENTES DE CASCABEL DE MONTAÑA(Crotalus aquilus, C. lepidus klauberi y C. lepidusxaquilus). PRESENTA Eric Abdel Rivas Mercado PARA OBTENER EL GRADO DE MAESTRÍA EN CIENCIAS; ÁREA DE TOXICOLOGÍA TUTOR (ES) Dr. en C. Eduardo de la Cerda González COMITÉ TUTORAL Dr. en C. Francisco Anibal Posadas del Río M. en C. José Luis Moreno Hernández Duque Aguascalientes, Ags, Noviembre de 2014. AGRADECIMIENTOS. A mis padres: Lic. José Reyes Rivas Enríquez y Bertha Alicia Mercado Romo por su esfuerzo e incondicional apoyo brindado. A mi novia: Kattia Estefanía Aguirre Ramírez, por brindarme un apoyo integral, durante la elaboración de este trabajo. Al Consejo Nacional de Ciencia y Tecnología (CONACYT) por brindar beca de manutención durante el tiempo en que se realizó este trabajo. A mi comité de tutores: Dr. en C. Eduardo de la Cerda González, Dr. en C. Francisco Anibal Posadas del Río y M. en C. José Luis Moreno Hernández Duque. Por su importante colaboración para los trabajos de campo y colecta a SEMARNAT, por brindar el permiso de colecta de los organismos. A mi grupo de apoyo de campo: M en C. Rubén Alonso Carbajal Márquez, Biol. Marco Antonio Domínguez de la Riva, Biol. Juan Daniel Rendón Trinidad, Kattia Estefanía Aguirre Ramírez. Al Instituto de Biotecnología de la Universidad Nacional Autónoma de México, Laboratorio del Dr. Alejandro Alagón Cano, Investigador Titular “C”. M. en C. Melisa Bénard Valle y M. en C. Edgar Neri, ayudante de investigador Instituto de Biotecnología, UNAM. Por brindarme la posibilidad de llevar a cabo dos estancias de investigación en los laboratorios del IBt UNAM. ÍNDICE GENERAL. ÍNDICE GENERAL. ............................................................................................................ 1 ÍNDICE DE TABLAS. ......................................................................................................... 4 ÍNDICE DE FIGURAS. ....................................................................................................... 5 ACRÓNIMOS. ...................................................................................................................... 6 RESUMEN ............................................................................................................................ 8 ABSTRACT. .......................................................................................................................... 9 INTRODUCCIÓN. ............................................................................................................. 10 1. Clasificación taxonómica de los reptiles. ...................................................................... 12 2. Reptiles venenosos. .......................................................................................................... 13 3. Aparato inoculador de veneno. ...................................................................................... 14 3.1 Serpientes venenosas en México. ................................................................................ 15 3.2 Serpientes venenosas en el estado de Aguascalientes. ................................................ 15 4. Organismos de estudio. ................................................................................................... 16 4.1 Crotalus aquilus (Klauber, 1952). .............................................................................. 16 4.2 Crotalus lepidus klauberi (Kennicott, 1861). ............................................................. 17 4.3 Crotalus lepidus x aquilus (Bryson R. W., 2010). ................................................. 19 5. Caso clínico. .................................................................................................................. 19 6. Veneno de serpientes. ..................................................................................................... 22 6.1 Características del veneno de serpientes y sus efectos. ............................................... 22 6.2 Principales componentes del veneno de serpientes. .................................................... 23 Metaloproteasas (SVMP’s). .......................................................................................... 24 Serin proteasas tipo trombina (SVSPs). ........................................................................ 25 Nucleasas, nucleotidasas y fosfomonoesterasas. ........................................................... 25 Fosfolipasas A2. ............................................................................................................. 26 Acetilcolinesterasa. ........................................................................................................ 27 L-Aminoácido oxidasas. ................................................................................................ 28 1 Agentes anti – agregación plaquetaria. .......................................................................... 29 6.3 Toxinas como agentes terapéuticos. ............................................................................ 30 Agentes pro y anti – coagulantes. .................................................................................. 34 Agentes anti – cáncer. .................................................................................................... 34 Desintegrinas. ................................................................................................................ 35 Crotoxina. ...................................................................................................................... 37 7. Hipótesis. .......................................................................................................................... 39 8. Ojetivos. ........................................................................................................................... 39 8.1 Objetivo general. ......................................................................................................... 39 8.2 Objetivos particulares. ................................................................................................. 39 9. Materiales y metodología. .............................................................................................. 40 9.1 Trabajo de campo; colecta de los ejemplares de víbora de cascabel. ......................... 40 9.2 Extracción y liofilización del veneno. ......................................................................... 41 9.3 Cuantificación de proteína por el método del ácido bicinconínico (BCA). ................ 41 9.4 Cuantificación de proteína por absorbancia a 280 nm. ............................................... 42 9.5 Electroforesis en gel de poliacrilamida en presencia de Dodecil sulfato sódico (SDS – PAGE). .............................................................................................................................. 42 9.6. Cromatografía líquida de alta resolución, fase reversa (RP-HPLC). ......................... 43 9.7Dosis letal media (DL50). ............................................................................................. 44 9.8 Actividad procoagulante. ............................................................................................ 45 9.9Actividad proteolítica sobre azocaseína. ...................................................................... 45 9.10 Detección de crotoxina: Western blot semi – seco. .................................................. 46 9.11 Detección y cuantificación de crotoxina mediante ELISA. ...................................... 47 10. Resultados. ..................................................................................................................... 49 10.1 Trabajo de campo; colecta de los ejemplares de víboras de cascabel. ...................... 49 10.2 Extracción, liofilización y cuantificación de proteína del veneno de cada serpiente, peso en mg/ordeña, BCA y absorbancia a 280nm. ........................................................... 49 10.3 Electroforesis en gel de poliacrilamida en presencia de dodecil sulfato sódico (SDS - PAGE). .............................................................................................................................. 50 10.4 Cromatografía líquida de alta resolución, fase reversa (RP-HPLC). ........................ 52 2 10.5 Dosis letal media (DL50). .......................................................................................... 54 10.6 Actividad pro coagulante. ......................................................................................... 55 10.7 Actividad proteolítica sobre azocaseína. ................................................................... 56 10.8 Detección de Crotoxina por RP – HPLC. ................................................................. 57 10.9 Detección de crotoxina: Western blot semi – seco. .................................................. 58 10.10 Detección y cuantificación de Crotoxina mediante ELISA. ................................... 59 11. Discusiones. .................................................................................................................... 60 Letalidad. ....................................................................................................................... 62 Actividad biológica. ...................................................................................................... 64 CONCLUSIÓN. .................................................................................................................
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